The overall objective of this subproject is to elucidate the molecular mechanism(s) whereby Francisella tularensis activates immune effector cells, a sentinel event in the early host response. Lipoproteins, beta-glucan, and LPS are the principle constituents of the F. tularensis cell wall. In other bacterial pathogens, these molecules are endowed with potent immunobiological properties many of which are mediated through interaction with pattern recognition receptors (PRR), such as CD14 and toll-like receptor 2 (TLR2), on the host cell surface. Very little is known, however, regarding the potential of francisellae cell wall constituents to activate immune effector cells and thus contribute to tularemia pathogenesis. Studies detailed herein will fill this critical gap in our understanding of the mechanism(s) whereby F. tularensis causes disease. Using the live vaccine strain of F. tularensis and CD14-deficient C3H/HeN mice we have begun to explore the possibility that francisellae stimulate host responses via binding to PRR. Based upon our findings, we hypothesize that F. tularensis lipoproteins/beta-glucans engage the CD14-TLR2 signaling pathway in innate immune cells thus triggering Th1-type host responses that engender the clinical manifestations of tularemia. This hypothesis will be tested through pursuit of the following Specific Aims: 1) compare the immunobiological activities of lipoproteins, beta-glucan, and lipopolysaccharide (LPS) isolated from F. tularensis, 2) determine the role of CD14 and TLR2 in macrophage activation by lipoproteins, beta-glucan, and LPS isolated from F. tularensis, and 3) determine the role of CD14 and TLR2 during primary pneumonic infection with F. tularensis. It is our expectation that the experimental approach detailed herein will identify the F. tularensis component(s) responsible for immune cell activation and elucidate the molecular mechanism(s) by which this event occurs. The rationale underlying this work is the belief that defining immune mechanisms that engender clinical manifestations and disease resolution is an essential first step towards development of immunotherapeutic strategies and identification of appropriate vaccine targets.